Sequence Characterized Amplified Region Markers Research Articles

Molecular Identification and Scar Detection of Entomopathogenic Fungi of the Genus Beauveria

In recent years, there has been increasing interest in identifying and studying beneficial microorganisms that can be used in organic farming as fertilizers and plant protection agents. Entomopathogenic fungi with an endophytic nature of the genus Beauveria are quite promising and their identification and detection in the plant and soil samples are of great importance. Molecular identification of fungi of the genus Beauveria was conducted by sequencing the internal transcribed spacer 1 and 5.8S ribosomal RNA gene (ITS) regions with universal primers. As a result of the sequencing, it was found that the analysed strains belong to four genera of Beauveria with the following percentage distribution: B. bassiana (68.18%), B. pseudobassiana (13.64%), B. varroae (9.09%) and B. tenella (9.09%). The largest percentages of the strains were identified as B. bassiana. Sequence-characterized amplified regions (SCAR) have been applied for the detection of Beauveria strains in the field samples. The efficiency and ability of three pairs of SCAR markers to differentiate the studied strains were tested. The applied SCAR markers generated fragments with expected length in more than 90% of the investigated strains and could be used as markers for the detection of the fungi from the genus Beauveria in field samples. In the selected experimental conditions only B. varroe 501 and B. tenella 593 lack fragments with used SCAR markers.

Morphological and Molecular Identification of Root-knot Nematode, Meloidogyne incognita Infecting Pomegranate (Punica granatum L.) in Jodhpur, Rajasthan, India

Root-knot nematodes (Meloidogyne spp.) pose a major threat to pomegranate (Punica granatum L.) cultivation in India, leading to significant yield losses. In this study, galled roots of pomegranate were collected from orchards in Jodhpur, Rajasthan and nematode infestation was confirmed through root staining. The perineal pattern of females displayed the typical characteristics of the Meloidogyne incognita. To further confirm the species, DNA was extracted and a polymerase chain reaction (PCR) assay using species-specific SCAR (sequence-characterized amplified region) primers was conducted. The M. incognita- species specific primers, MincF1/MincR1, yielded the expected 150 bp product, verifying the presence of M. incognita. This study marks the first molecular confirmation of M. incognita infecting pomegranate orchards in this region, demonstrating the efficacy of SCAR markers in complementing traditional morphological identification as well.

The development and utilization of two SCAR markers linked to the resistance of banana (Musa spp. AAA) to Fusarium oxysporum f. sp. cubense race 4

Banana (Musa spp.) production worldwide is seriously threatened by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). The best way to control this disease is to grow resistant cultivars. However, it requires large-scale field evaluations and labor- and time-consuming to obtain disease-resistant germplasm. Development of early, reliable, and reproducible selection strategies are considered as the efficient approach which could speed up the selection procedure. In this study, two pairs of sequence-related amplified polymorphism (SRAP) primers related to banana Foc resistance/susceptibility were screened from 100 pairs of random primers. Correspondingly, two pairs of sequence characterized amplified region (SCAR) markers (namely SC4-F/SC4-R and SC14-F/SC14-R, respectively) were successfully generated from these two SRAP markers using 30 cultivars either resistant or susceptible to Foc. Both SCAR markers were located in mitochondrion genome and showed discriminatory power of 96.67% and 100%, respectively. Mitochondrial proteins possibly play a very important role in banana resistance to Foc. In additional, these two SCAR markers were employed simultaneously to screen potential resistant germplasm from 53 accessions with unknown resistance to Foc, and the results revealed a consistency of 83.0% with each other, further indicating their high reliability and reproducibility. These results suggest that both SCAR markers could be used in molecular marker-assisted selection for banana germplasm resistant to Fusarium.

Development of highly discriminatory SCoT- and CBDP-based SCAR fingerprint for authentication of Indian senna (Senna alexandrina Mill.) formerly Cassia angustifolia Vahl.).

Indian senna (Senna alexandrina Mill.) (formerly Cassia angustifolia Vahl.) is an important medicinal plant of the family Fabaceae. The leaves and pods of Indian senna yield sennosides and rhein-based laxative. Adulteration of Indian senna is a serious issue as with most of the medicinal plants used in the Indian systems of traditional medicine. The bulk of dried leaves and pods of morphologically related species, such as Cassia fistula, Senna occidentalis, Senna sophera, and Senna tora, is usually mixed with those of the Indian senna, and the admixture is used in laxative-based formulations. The present investigation is a modest attempt at developing species-specific start codon targeted (SCoT) polymorphism- and CAAT-box-derived polymorphism (CBDP)-based sequence-characterized amplified region (SCAR) markers for the identification and authentication of Indian senna and four adulterant species (C. fistula, S. occidentalis, S. sophera, and S. tora species). In this study, genomic DNA extracted from 44 accessions of Indian senna and four adulterant species was subjected to SCoT and CBDP PCR. The polymorphic amplicons were identified, eluted, ligated, and transformed into Escherichia coli DH5 α strain. PCR, restriction analysis, and DNA sequencing confirmed the transformed recombinant plasmid clones. Post-sequencing, the sequence of the primary SCoT and CBDP primers was analyzed and extended into the unique signature sequence of the concerned accessions. This resulted in development of one SCoT-44- and two CBDP-25-based SCARs. SCoT-44 SCAR produced a signature amplicon of 287 bp for accession DCA120, and CBDP-25 SCAR yielded signature amplicons of 575 and 345 bp for accessions DCA13 and DCA119, respectively. The developed SCAR markers were validated across 48 samples (44 accessions of Indian senna and 4 adulterant species) and produced distinct amplicons in Indian senna only, while no such amplicon was observed in the other four adulterant species. The information generated using these markers have been faithfully converted to single-locus, unequivocal, highly reproducible, and informative sequence-based SCAR markers. These markers will enable discrimination of individual plants on the basis of unique sequence-specific amplicons, which could be used as diagnostic markers to settle issues pertaining to the true identity of Indian senna.

Discrimination of lethal Russula subnigricans from wild edible and morphologically similar mushrooms in the genus Russula using SCAR markers

Russula is a diverse genus of mushrooms that is consumed worldwide. Russula subnigricans is a lethal species that causes myotoxicity upon ingestion. It is morphologically similar to wild foraged species, including R. nigricans and R. densifolia. Misidentification of these mushrooms can lead to severe food poisoning. Several PCR-based approaches have been used to overcome this problem. In this study, 22 samples of wild edible Russula species were collected, and five clinical samples of R. subnigricans leftovers were obtained. Based on the sequence data of the internal transcribed spacer, the phylogenetic trees revealed that these 27 samples could be classified into well-established clades of six subgenera. Fifty arbitrary primers were used in high annealing temperature rapid amplified polymorphic DNA (HAT-RAPD) technique to screen polymorphic bands among R. subnigricans, R. nigricans, R. densifolia, and other edible species. Distinctive RAPD fragments were obtained from 11 primers and used in sequence characterized amplified region (SCAR) technique. Three pairs of species-specific SCAR primers were selected to distinguish lethal R. subnigricans, R. nigricans, and R. densifolia with SCAR markers of 213, 420, and 609 bp, respectively. Ten-fold sensitivity test revealed that the SCAR marker of R. subnigricans could be detected at DNA template concentrations as low as 30 pg/μL. This marker could detect the presence of lethal R. subnigricans even at 1% in a cooked sample of mixed mushroom remnants. The developed SCAR markers ensured rapid and accurate diagnosis of suspected myotoxic mushrooms, thereby reducing the risk of severe foodborne illness and death.

Characterization of Trichoderma strains for novel species-specific markers by multiplex PCR and antagonistic property against Alternaria ricini in castor (Ricinus communis L.)

The study aimed at characterizing six Trichoderma strains isolated from the rhizospheric soil of castor growing region of Telangana, India. Microscopic examination and sequencing of ribosomal DNA (rDNA) using internal transcribed spacer (ITS) and D1-D2 region-specific primers revealed them as members of Trichoderma sp. Whereas the phylogenetic analysis revealed four of those strains as Trichoderma asperellum, two strains were confirmed as Trichoderma harzianum with a high sequence similarity of >99%. Further, multiplex PCR results with Trichoderma-specific primers; TEF1 (translation elongation factor 1) and rpb2 (the second largest subunit of RNA polymerase II) gene-specific primers confirmed the findings resulting from the phylogenetic analysis. To develop a set of sequence characterized amplified region (SCAR) markers, these strains were screened with 185 random amplified polymorphic DNA (RAPD) primers. A pairwise comparison of correlation of coefficient values determined from the RAPD-based genetic similarity analysis revealed that TV3 and TS4 strains were closely related to each other (0.98) confirming them as T. harzianum while N13, 7316, TV5, and TV2B16 strains formed a different cluster with T. asperellum strains. A set of novel SCAR markers were developed to distinguish these two species, T. asperellum and T. harzianum including the reported Trichoderma strains as Ex-type strains. T. harzianum strains had extremely low sporulation ability to be tested for biocontrol properties. Of the four T. asperellum strains, 7316 followed by N13 demonstrated high antagonistic ability against the fungal pathogen, Alternaria ricini indicating these strains as potential biocontrol agents to limit Alternaria infection in castor.

DNA barcodes, ISSR, RAPD and SCAR markers as potential quality control tools for molecular authentication of black and white mulberry

Morus species, especially black mulberry (Morus nigra L.), are well known for their nutritive and economic values. Black mulberry is often a subject for deliberate and undeliberate substitution with the morphologically similar white mulberry (Morus alba L.). In the current study, the genetic diversity of Morus nigra L. and Morus alba L. was assessed using different genetic profiling and fingerprinting techniques namely namely DNA barcoding, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). DNA barcoding was implemented using 2 plastid loci (matK, and rbcL-1) and 2 nuclear spacers (ITS and ITS2) aiming at accurate authentication of the studied species. DNA barcoding, targeting the selected loci succeeded to identify and authenticate the studies Morus species but failed to differentiate them, owing to their close genetic resemblance. Out of 52 screened RAPD primers, 27 were able to produce reproducible and clear profiles with 115 amplified fragments of which 37 were polymorphic accounting for 32.17% polymorphism. The number of amplified bands varied from 2 to 7 ranging in size from 100 to 700 bp. Moreover, 55 ISSR primers were evaluated, 108 fragments were produced, using 20 primers, of which 28 were polymorphic accounting for 25.92% polymorphism with size range of 200–800 bp. Sequence Characterized Amplified Region (SCAR) marker was developed based on reproducible banding pattern obtained by RAPD primer (OPG-03), for the purpose of accelerating the initial screening of Morus batches and enabling the preventative rejection of adulterated samples. The polymorphic band obtained was sequenced and specific primers were designed to develop a SCAR marker of approximately 558 bp exclusively for M. nigra, allowing detection of down to 1 ng, of M. nigra. The SCAR marker showed 100% success in differentiating authentic M. nigra and M. alba samples. In addition, the application of the developed SCAR marker was extended to distinguish M. nigra from its four common adulterants namely Ficus carica L., Fragaria vesca L., Ficus racemosa L. and Vitis vinifera L.. The proposed protocol was successfully implemented for the evaluation of 47 commercial M. nigra samples collected from different Egyptian locations.

Molecular cloning and development of RAPD-SCAR markers for the selection of thermo-tolerant line of tropical tasar silkworm

Aim: To develop random amplified polymorphic DNA (RAPD) -sequence-characterized amplified region (SCAR) markers for selecting thermo-tolerant line of tropical tasar silkworm. Methodology: In the environmental chamber, Daba BV cocoons of A. mylitta were exposed to high temperature at 46°C/4 hr for 3 days. After the emergence, genomic DNA was extracted from thermo-tolerant moths and thermo-susceptible pupae. The genomic DNA was amplified using 30 RAPD random decamer primers. The improved RAPD fragments that can differentiate thermo-tolerant and susceptible line of A. mylitta were eluted, cloned in pJET1.2 vector and sequenced. SCAR markers were developed based on the sequence and validated in the subsequent generations. Results: Among 30 RAPD primers, OPK04, OPAJ15 and OPA17 generated polymorphic bands to differentiate thermo-tolerant and susceptible line of A. mylitta. These polymorphic bands were eluted, cloned and sequenced. Sequencing of three cloned fragments revealed that clone PB1 comprised of 1412 bp, clone PB2 comprised of 704 bp and clone PB3 comprised of 931 bp. Sequence specific stable multiplex SCAR markers TT-PB1, TT-PB2 and TT-PB3 were designed and synthesized. PCR amplification was performed using DNA templates of 10 thermo-tolerant and 10 thermo-susceptible samples. SCAR marker TT-PB1 was observed to be more specific to thermo-tolerant line of tropical tasar silkworm and validation with 25 samples each in next generation also supported the specificity of TT-PB1. Interpretation: Among three SCAR markers, TT-PB1 showed more specificity for selecting the thermo-tolerant line of A. mylitta. Therefore, the study provides an effective and precise PCR-based molecular marker system for selecting thermo-tolerant lines in tropical tasar silkworm to overcome seed crop loss due to high temperature stress in tasar rearing hotter zones. Key words: Antheraea mylitta, cloning, RAPD, SCAR m­­­arker, Thermo-tolerance

A KASP Marker for the Potato Late Blight Resistance Gene RB/Rpi-blb1

The disease late blight is a threat to potato production worldwide, making genetic resistance an important target for breeding. The resistance gene RB/Rpi-blb1 is effective against most strains of the causal pathogen, Phytophthora infestans. Until now, potato breeders have utilized a Sequence Characterized Amplified Region (SCAR) marker to screen for RB. Our objective was to design and validate a Kompetitive Allele Specific PCR (KASP) marker, which has advantages for high-throughput screening. First, the accuracy of the SCAR marker was confirmed in two segregating tetraploid populations. Then, using whole genome sequencing data for two RB-positive segregants and a diverse set of 23 RB-negative varieties, a SNP in the 5’ untranslated (UTR) region was identified as unique to RB. The KASP marker based on this SNP, which had 100% accuracy in the cultivated diversity panel, was used to generate diploid breeding lines containing RB. The KASP marker is publicly available for others to utilize.

Genotyping-driven diversity assessment of biocontrol potent Bacillus spp. strain collection as a potential method for the development of strain-specific biomarkers.

Bacillus species are among the most researched and frequently applied biocontrol agents. To estimate their potential as environmentally friendly microbial-based products, reliable and rapid plant colonization monitoring methods are essential. We evaluated repetitive element-based (rep) and Random Amplified Polymorphic DNA (RAPD) PCR (Polymerase Chain Reaction) genotyping in a diversity assessment of 251 strains from bulk soil, straw, and manure samples across Serbia, highlighting their discriminative force and the presence of unique bands. RAPD 272, OPG 5, and (GTG)5 primers were most potent in revealing the high diversity of a sizable environmental Bacillus spp. collection. RAPD 272 also amplified a unique band for a proven biocontrol strain, opening the possibility of Sequence Characterized Amplified Region (SCAR) marker design. That will enable colonization studies using the SCAR marker for its specific detection. This study provides a guide for primer selection for diversity and monitoring studies of environmental Bacillus spp. isolates.

Development of sequence-characterized amplified region (SCAR) markers for accurate and differential identification of multienzyme-producing and non-enzymatic Aspergillus strains of industrial importance.

Aspergillus strains are known to produce multiple enzymes of industrial importance. To screenAspergillusisolates and select a strain with the ability to produce multiple enzymes and discriminate it from non-enzymatic strains, a rapid and accurate approach is required. With this background, a DNA fingerprinting-based study was conducted to develop a simple but accurate molecular detection method with the potential to discriminate multienzyme-producing Aspergillus strains from non-enzymatic strains, irrespective of species. To achieve this, Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR was employed to derive group-specific Sequence Characterized Amplified Region (SCAR) markers (i.e., markers corresponding to PCR amplicons of known DNA sequence). To this end, both group-specific (multienzyme-producing and non-enzymaticAspergillusgroup) SCAR markers were sought by comparing the ERIC fingerprint profiles and used to develop primers for use in specific and differential identification of multienzyme-producingAspergillusisolates. As an outcome, the two SCAR-PCR formats were developed. One format is for specific identification of multienzyme-producingAspergillusstrains (SCAR-PCR1), and the other for identifying non-enzymaticAspergillusstrains (SCAR-PCR2). Both SCAR-PCRs were able to discriminate between these two contrasting groups. These formats are simple but accurate and rapid compared to the time-consuming and laborious conventional methods. Therefore, they could be efficient as an alternative strategy for the high-throughput screening of industrially importantAspergillusstrains.

Development of real-time PCR and droplet digital PCR based marker for the detection of Tilletia caries inciting common bunt of wheat.

This is the first study reporting droplet digital PCR and quantitative real time PCR for detection of Tilletia caries (syn. T. tritici), which causes common bunt of wheat and leads to yield losses of 80% in many wheat growing areas worldwide. To establish an accurate, rapid and quantifiable detection method, we tested 100 inter simple sequence repeats (ISSR) primers and obtained a species-specific fragment (515 bp) generated by ISSR 827. Then, a specific 266 bp band for the sequence characterized amplified region (SCAR) marker was produced from T. caries. The detection limit reached 50 pg/μL. Based on the SCAR marker, we further developed a higher sensitivity of quantitative real time-polymerase chain reaction (qRT-PCR) with a detection limit of 2.4 fg/μL, and droplet digital PCR (ddPCR) with a detection limit of 0.24 fg/μL. Both methods greatly improved the detection sensitivity of T. caries, which will be contribute a lot for quickly and accurately detection of T. caries, which causes wheat common bunt.

Mendelian segregation for parthenogenetic embryo development at the diploid level in the flowering plant Erigeron.

Parthenogenesis is the capacity of organisms to develop embryos from unfertilized eggs. When parthenogenesis is coupled with unreduced gamete formation (apomeiosis), genetically maternal progeny result. Genetic elucidation of this form of reproduction in plants, apomixis, has important agronomic implications. However, genetic characterization of apomeiosis and parthenogenesis has been problematic in part because the traits usually co-occur and are restricted to polyploids. In this work, the inheritance of parthenogenetic embryo development, by itself, was studied at the diploid level. Progeny resulting from a cross between a diploid (2n = 18), heterozygous, parthenogenetic pollen donor, and a diploid, wildtype, sexual seed parent were evaluated. Paternity was tested with conserved orthologous sequence (COS) markers, reproductive development of F1s was evaluated with microscopy of cleared ovules, and an amplified fragment length polymorphism (AFLP) marker (Eagc × Macg.615) co-segregating with parthenogenesis was characterized at the sequence level. Of 102 diploid biparental progeny, 47 exhibited parthenogenetic embryo and endosperm development, and 55 lacked development of the egg and central cell. This result is consistent with Mendelian inheritance for a single locus (P = 0.43). Isolation and sequencing of the AFLP marker indicates that it is likely a portion of a Ty-Gypsy retrotransposon. Attempts to develop a sequence-characterized amplified region marker from the AFLP were unsuccessful. This work shows that parthenogenesis can be transmitted simply at the diploid level. This advance is key in the development of a tractable system in Erigeron aimed at the identification of the parthenogenesis locus using genetic mapping strategies.

SCAR marker: A potential tool for authentication of agriculturally important microorganisms.

Microbial inoculants are globally recommended for plant growth promotion and control of plant pathogens. These inoculants require stringent quality checks for sustainable field efficacy. Questionable regulatory frameworks constantly deteriorate the reliability of bio-inoculant technology. Existing global regulations do not involve any rapid molecular technique for the routine inspection of microbial preparations. Sequence characterized amplified region (SCAR) marker offers rapid and precise strain-level authentication of target microbes. Such advanced molecular techniques must be exploited to accurately validate the microbial formulations. Besides, the global dissemination of plant pathogenic microbes has always been an alarming threat to food security. SCAR markers could be used at the plant quarantine centers to rapidly detect catastrophic pathogens, thereby circumventing the import and export of contagious plant materials. The current review is focused on promoting the SCAR marker technology to validate commercial bio-inoculants and predict plant pandemics.

Validation of PCR-based Markers Associated with Sex Determination in Date Palm (Phoenix dactylifera L.)

Phoenix dactylifera (Date palm) is one of the essential commercial and economical fruit crops which mainly grows in hot arid areas. A good source of food, shelter, and energy, date palm is widely known due to its unique characteristics. The dioecious nature of date palm made it challenging to prognosticate its sex until they flower almost 5 to 8 years after plantation. Researchers have developed many strategies to determine sex, including morphological study, biochemical studies, molecular-based studies as sequence characterized amplified regions (SCAR), randomly amplified polymorphic DNA (RAPD) marker, and polymerase chain reaction (PCR) assays, but still more endeavours are integral. To foretell sex at the seedling stage, specific PCR primers were employed in this study. These primers were designed on sex-linked regions that determine their gender. Date palm DNA was extracted through the CTAB method. The DNA was then amplified through PCR using sex-linked specific primers. PCR products have run on gel electrophoresis using 1.2% agarose to score the bands for sex determination. Four primers were employed in this study to determine sex. Two primers were SCAR markers, and one was a sex-linked tetra primer. Tetra-primer amplified the double band fragment of 430 and 320bp respectively in male plants, while a band of 430bp was amplified in female plants. The other primer named ALAMER1 was amplified by the single band fragment of 186bp in males with no band in females. The fourth primer, named as SCARdp, amplified the band fragment of 354bp fragment in males. These SCAR markers were established to be male-specific. Sex-linked markers provide a platform for robust, efficient, and accurate determination of sex in date palms.

Genetic relationship and species identification of Dioscorea polystachya Turcz. in Yams determined by ISSR, ISAP, SRAP and SCAR markers

Dioscorea polystachya Turcz. is the authentic herb Yam in Chinese traditional medicine. In the medicinal marketplace, some related Dioscorea species are mislabeled as “Yams”, such as D. alata L., D. exalata C.T. Ting & M.C. Chang, D. persimili Prain et Burkill, D. fordii Prain et Burkill and D. japonica Thunb. To determine the genetic relationships among these species and develop a rapid and accurate method for D. polystachya identification, Inter Simple Sequence Repeat (ISSR), Intron Sequence Amplified Polymorphism (ISAP), Sequence-Related Amplified Polymorphism (SRAP) and Sequence Characterized Amplified Region (SCAR) markers were used in this study. By combining ISSR, ISAP and SRAP markers, a high degree of variation among the Dioscorea species was detected. Moreover, a relatively high gene flow (Nm = 0.3293) among the species was observed. This may result from gene pool exchanges of wild materials. The phylogenetic tree and genetic similarity analysis showed that D. japonica had the closest genetic relationship with D. polystachya. Considering the lowest inter-species variation observed in D. polystachya and the highest genetic divergence within D. japonica, we suggest a taxonomy of a one-species complex, D. polystachya–D. japonica, for breeding and conservation purposes. The phylogenetic data in this study indicated that D. alata and D. persimilis are independent species, not cultigens. Furthermore, a SCAR marker was developed that could be used to discriminate the authentic herb D. polystachya from other Dioscorea species. Overall, these results are useful as guidelines for breeding programs and conservation actions.

Development of SCAR Markers to Identify Medicinal Cultivars of Paeonia lactiflora.

Paeoniae Radix, the dried root of Paeonia lactiflora, is one of the most important ingredients in Kampo medicine. It is known that Paeoniae Radix is derived from various P. lactiflora cultivars, including medicinal and horticultural cultivars, and that cultivar identification by DNA analysis has been unsuccessful. We attempted to develop sequence characterized amplified region (SCAR) markers as useful DNA markers for the identification and herbal medicine authentication of two cultivars developed in Japan, 'Bonten' and 'Kitasaisho,' which are two superior medicinal strains of P. lactiflora. Sequence-related amplified polymorphism (SRAP) analysis was conducted on fourteen P. lactiflora cultivars, and polymorphic fragments specific to 'Bonten' or 'Kitasaisho' were detected. Then, SCAR markers for 'Bonten' and 'Kitasaisho' were developed from the sequence information of these polymorphic fragments. Thirty cultivars of P. lactiflora and five herbal medicine samples were used to validate the specificity of the developed SCAR markers. As a result, we confirmed that our SCAR markers can identify 'Bonten' or 'Kitasaisho' from the plant samples and the herbal medicine samples. Thus, we have successfully designed two highly specific DNA markers and established an easy, rapid, and cost-efficient method to identify specific cultivars of P. lactiflora. Our SCAR markers are expected to contribute to the maintenance of P. lactiflora cultivars such as 'Bonten' as superior medicinal strains, the development of more elite cultivars in the future, and the deterrence of outflow of original cultivars to foreign countries.

Development and Application of a Cultivar-Specific Sequence-Characterized Amplified Region (SCAR) Marker for the Detection of Chrysanthemum morifolium Ramat. 'Daboju'.

Chrysanthemummorifolium Ramat. ‘Daboju’ is a C. morifolium cultivar with important ornamental and medicinal values, and is often used in the treatment of colds, blurred vision, dizziness, and itchy skin. As the morphological characteristics of C. morifolium ‘Daboju’ are very similar to those of other C. morifolium cultivars, they are often confused in practice. However, the medicinal value and practical use of C. morifolium depends on using the correct rapid and accurate identification of C. morifolium ‘Daboju’ and its differentiation from other, morphologically similar C. × morifolium cultivars. Twenty-one polymorphic start codon-targeted (SCoT) primers were amplified in 21 distinct C. morifolium cultivars. One cultivar-specific DNA marker was developed with the aim of the rapid and accurate identification of C. morifolium ‘Daboju’ and its differentiation from other, similar C. morifolium cultivars. Twenty-one polymorphic start codon-targeted (SCoT) primers were amplified in 21 distinct C. morifolium cultivars. One cultivar-specific 385-bp amplicon (named SCoT36-385), amplified only in C. morifolium ‘Daboju’ (and in all samples of this cultivar), was identified, cloned, and sequenced. Subsequently, a sequence-characterized amplified region (SCAR) marker (named DBJF/DBJR), generating a 360-bp amplicon, was developed from SCoT36-385 and tested for amplification in all 21 C. morifolium cultivars, ten C. morifolium ‘Daboju’ populations, and different simulated adulterations of ‘Daboju’ with other cultivars. The primers amplified the specific 360-bp-long DNA fragment in all the tested C. morifolium ‘Daboju’ samples but failed in the absence of ‘Daboju’. The detection limit of the SCAR primer pair (DBJF/DBJR) was 100 pg of DNA extracted from C. morifolium ‘Daboju’. Hence, this SCAR marker has a very high detection sensitivity, and can be used for accurate and rapid identification of C. morifolium ‘Daboju’. It can play an important role in ensuring the quality of medicinal preparations and protecting C. morifolium ‘Daboju’ germplasm resources in breeding programs and in identifying lines generated from this cultivar.

Distribution of Puccinia striiformis f. sp. tritici Races and Virulence in Wheat Growing Regions of Kenya from 1970 to 2014.

Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici, is a major threat to wheat (Triticum spp.) production worldwide. The objective of this study was to determine the virulence of P. striiformis f. sp. tritici races prevalent in the main wheat growing regions of Kenya, which includes Mt. Kenya, Eastern Kenya, and the Rift Valley (Central, Southern, and Northern Rift). Fifty P. striiformis f. sp. tritici isolates collected from 1970 to 1992 and from 2009 to 2014 were virulence phenotyped with stripe rust differential sets, and 45 isolates were genotyped with sequence characterized amplified region (SCAR) markers to differentiate the isolates and identify aggressive strains PstS1 and PstS2. Virulence corresponding to stripe rust resistance genes Yr1, Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, and Yr27 and the seedling resistance in genotype Avocet S were detected. Ten races were detected in the P. striiformis f. sp. tritici samples obtained from 1970 to 1992, and three additional races were detected from 2009 to 2014, with a single race being detected in both periods. The SCAR markers detected both Pst1 and Pst2 strains in the collection. Increasing P. striiformis f. sp. tritici virulence was found in the Kenyan P. striiformis f. sp. tritici population, and different P. striiformis f. sp. tritici race groups were found to dominate different wheat growing regions. Moreover, recent P. striiformis f. sp. tritici races in East Africa indicated possible migration of some race groups into Kenya from other regions. This study is important in elucidating P. striiformis f. sp. tritici evolution and virulence diversity and useful in breeding wheat cultivars with effective resistance to stripe rust.

Identification of resistance sources in common bean (

Context Among biotic factors, bean anthracnose caused by Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara is one of the most destructive diseases of common bean (Phaseolus vulgaris L.). Resistance in common bean genotypes is controlled by anthracnose resistance loci (designated Co). Aims The objective of this study was to evaluate the presence of Co resistance genes in common bean genotypes from Turkey and to examine promising genotypes as candidates for parent plants in breeding studies. Methods The presence of Co resistance genes in 693 common bean genotypes from 35 provinces of Turkey was determined by 12 SCAR (sequence characterised amplified region), RAPD (random amplified polymorphic DNA), STS (sequence-tagged site) and CAPS (cleaved amplified polymorphic sequence) markers. The disease reaction of 40 agronomically promising genotypes to different pathogen isolates was evaluated in a pot study. Key results The results indicated the presence of Co-1, Co-14, Co-15, Co-2, Co-33, Co-4, Co-42, Co-6, Co-10, Co-11 and Co-13 resistance genes, alone or in combination; Co-5 was not detected in any of the genotypes. Among the genotypes, Co-15 was the most frequent resistance source, followed by Co-4, Co-6 and Co-11, respectively. Common bean genotypes carrying Co-14, Co-2, Co-33, Co-11 and Co-13 have been identified for the first time in Turkish germplasm. Five genotypes carrying the various combinations of Co-1, Co-15, Co-4, Co-6, Co-10, Co-11 and Co-13 were resistant to all isolates in pathogenicity tests. Conclusions This study is the most comprehensive to date on Co genes linked to the resistance of common bean genotypes throughout Turkey and provides an important gene pool for breeding studies into bean anthracnose. Resistant genotypes identified will be useful as parent plants in resistance breeding studies. Implications Resistance genotypes carrying different Co genes to bean anthracnose will contribute significantly to the disease management strategy.